1. Field of the Invention
The invention relates generally to compositions and methods for facilitating the construction of recombinant nucleic acid molecules, and more specifically to compositions for using one or more topoisomerases to generate covalently closed recombinant nucleic acid molecules and to methods of making such recombinant nucleic acid molecules.
2. Background Information
The advent of recombinant DNA technology has allowed the cloning and identification of genes from many different organisms, and the determination of the complete genomes of an ever-increasing number of organisms, including humans. The elucidation of a large number of new and uncharacterized genes creates a pressing need for technologies that enable rapid expression and analysis of these genes. The ability to construct recombinant nucleic acid molecules has provided a means to produce novel “gene products” and to express gene products, particularly heterologous gene products, in cells, tissues and organisms in which they are not normally produced. Thus, recombinant DNA technology has led, for example, to the fields of gene therapy, in which defective genes are replaced by copies of a normal gene; and “biopharming,” in which, for example, a gene product such as an antibody, which normally is produced by an animal, is expressed in a plant, thereby allowing large scale production of the gene product.
Despite the great leaps in progress that have resulted from the discovery and development of recombinant DNA methods, a great number of steps often is required to prepare a novel DNA construct having desired properties. A significant bottleneck in recombinant DNA methodology is the requirement that each nucleic acid sequence that is to be used to prepare a construct must be cloned into a vector, the vector must be introduced into and amplified in a host cell (generally a bacterial cell), the amplified vector must be isolated from the host cell, and then must be transformed or transfected into the appropriate cell type for expression. Vectors with the appropriate functional elements such as a promoter, an origin of replication, a selectable marker, an epitope tag, or the like may need to be constructed. Such methods require multiple restriction enzyme digestion and ligation steps, in addition to numerous purification and characterization steps.
Methods and products are being developed to reduce the number of steps required to obtain a desired nucleic acid construct. For example, many commercial suppliers provide vectors that contain one or more functional elements of interest, and have cloning sites such that a desired nucleotide sequence can be cloned in frame with the sequences in the vector. However, such vectors are limited in that only the most commonly used elements such as particularly useful promoters or tags or the like can be included in the vectors in order for the vector to be commercially viable.
In some cases, there may be no need to covalently ligate together nucleic acid sequences that have been allowed to join. For example, non-covalently linked constructs formed by hybridization of complementary overhanging ends can be used to transfect cells with a reasonably high efficiency. However, such constructs effectively contain “nicks” at the sites of hybridization and, therefore, are more susceptible to endonuclease degradation than covalently linked sequences. Furthermore, constructs containing nicks are not suitable for certain further manipulations such as amplification by a polymerase chain reaction. Thus, a need exists to identify methods for facilitating the preparation of nucleic acid constructs. The present invention satisfies this need and provides additional advantages.